Wenyan Lai

Effects of Sex Hormones and Their Balance on the Proliferation of Rat Vascular Endothelial Cells

Objective: To investigate the adjustment of estrogen, progesterone and testosterone on the proliferation of female and male rat vascular endothelial cells (VECs) separately. Methods: Rat lung VECs were cultured according to the block explanting method. MTT assay was used to measure the proliferation of VECs. Results: 17β-Estradiol (E2) at 3 × 10–8 and 3 × 10–7 M accelerated the proliferation of female rat VECs (p < 0.01). E2 at 3 × 10–9, 3 × 10–8 and 3 × 10–7 M accelerated the proliferation of male rat VECs (p < 0.05). Tamoxifen, the estrogen receptor antagonist, could block the effect of estrogen on the proliferation of VECs. Testosterone at 3 × 10–8 and 3 × 10–7 M significantly increased the proliferation of male rat VECs (p < 0.05), but had no effect on female rat VECs. Progesterone at 10–9 and 10–8 M had no effect on female rat VECs alone. When the ratio of E2 to progesterone was 3/10, the proliferation of female rat VECs was accelerated (p < 0.05). When the ratio of E2 to testosterone was 1/1, the proliferation of female rat VECs was also hastened (p < 0.05). However, when the ratio was reduced to 1/100, the hastening effect disappeared. Conclusion: Estrogen can speed up the proliferation of female and male rat VECs, while progesterone has no effect on female rat VECs alone. The balance of the ratio of E2 to testosterone, E2 to progesterone may play an important role in the proliferation of female rat VECs.

Research on the age-related changes in the nitric oxide pathway in the arteries of rats and the intervention effect of dehydroepiandrosterone.

Background: Aging, especially the aging of blood vessels, is a dangerous independent factor in the occurrence and development of cardiovascular and cerebrovascular diseases. However, the cellular and molecular pathologic mechanisms underlying the aging of blood vessels remain unclear. Purpose: To observe changes in the nitric oxide (NO) pathway and the interventional effects of dehydroepiandrosterone (DHEA) in the artery during the aging process. Methods: Male Wistar rats were divided into 3 experimental groups of 6 rats each: group 1, adult control 12-month-old rats fed conventional fodder; group 2, 18-month-old rats fed conventional fodder, and group 3, 18-month-old rats that received 1 mg/kg/ DHEA in their fundamental fodder when they were 12 months old. The thoracic aorta was chosen. Endothelial NO synthase protein was measured using the Western blot method, the amount of NO using the Griess method, the amount of cGMP using radioimmunoassay, and the activity of superoxide dismutase (SOD) and the amount of malonyldialdehyde (MDA) using colorimetry. Result: Compared with the adult group, in the aortas of 18-month-old rats the protein expression of endothelial NO synthase (eNOS) is lower; the activity of SOD is lower but the amount of MDA is higher; the amounts of NO and cGMP are lower, but after DHEA intervention these changes are apparently ameliorated in the aged group. Conclusion: During the aging process in rat arteries, the expression of eNOS is lowered, the function of oxidation resistance is weakened, and the response of the vascular smooth muscle to NO is apparently decreased. DHEA is able to ameliorate the function of NO-related signal pathways and delay the aging process of the blood vessels.

Testosterone suppresses oxidative stress via androgen receptor-independent pathway in murine cardiomyocytes

Evidence supports that oxidative stress exerts significant effects on the pathogenesis of heart dysfunction. On the other hand, the presence of specific androgen receptor (AR) in mammalian cardiomyocytes implies that androgen plays a physiological role in cardiac function, myocardial injury and the regulation of the redox state in the heart. This study used the testicular feminized (Tfm) and castrated male mice to investigate the effects of testosterone deficiency, physiological testosterone therapy and AR on oxidative stress in cardiomyocytes. Tfm mice have a non-functional AR and reduced circulating testosterone levels. Male littermates and Tfm mice were separated into 5 experimental groups: non-castrated littermate controls, castrated littermates, sham-operated Tfm, testosterone-treated castrated littermates and testosterone-treated sham-operated Tfm mice. Cardiomyocytes that were isolated from the left ventricle were used for determination of superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px) enzyme activities, and malondialdehyde (MDA) levels. Additionally, mitochondrial DNA (mtDNA) deletion mutations were detected by nested PCR. The SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the proportion of mtDNA mutations were increased in castrated and sham-operated Tfm mice compared to control mice. However, an increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the proportion of mtDNA mutations in the mice that had received testosterone therapy. These changes were statistically similar in castrated and sham-operated Tfm mice after testosterone therapy. In conclusion, it is testosterone deficiency that induces oxidative stress in cardiomyocytes. Physiological testosterone therapy is able to suppress oxidative stress mediated via the AR-independent pathway.

Estrogen Receptors and the Activity of Nitric Oxide Synthase in the Artery of Female Rats Receiving Hormone Replacement Therapy

Objective: To observe the expression of estrogen receptor and the activity of NOS in the arteries of female rats receiving estrogen replacement therapy. Methods: Seventy-two female rats were randomly divided into four groups: group A: sham-ovariectomy; group B: ovariectomy; group C: ovariectomy with estrogen replacement therapy (benzoate estradiol, 5 μg i.m. once in 2 days); group D: ovariectomy with estrogen and progesterone replacement therapy (benzoate estradiol, 5 μg i.m. once in 2 days and progesterone, 1 mg i.m. once in 2 days). The rats were killed after 2 months. The receptor-binding assay was adopted to measure the estrogen receptors in the arteries of the rats, and the activity of NOS in the arteries was assessed by the hemoglobin reductase method. Results: The ER number and NOS activity in the arteries of the ovariectomized group are less than those in sham-ovariectomy group (p < 0.05). The ER number and NOS activity in the arteries of groups C and D are larger and higher than those in the ovariectomized group (p < 0.05). No significant differences in the ER number and NOS activity were observed between groups C and D. Conclusion: The ER number and NOS activity in the rat artery significantly decrease after ovariectomy, while hormone replacement therapy can significantly increase the artery NOS activity and retain the ER number in the artery of the ovariectomized rats to normal level. The result may contribute to explaining the beneficial effect of estrogen in the prevention of coronary artery diseases in postmenopausal women.

Heat shock protein 65 promotes atherosclerosis through impairing the properties of high density lipoprotein

AIM To explicit whether the functions of high density lipoprotein (HDL) are impaired in murine atherosclerosis by subcutaneous immunization with recombinant mycobacterial heat shock protein 65 (HSP65). METHODS C57BL/6 mice were fed a normal chow-diet with non immunization as normal group. ApoE knockout (ApoE(-/-)) mice on high-fat diet were randomly divided into three groups (n = 8) and immunized subcutaneously with different concentrations of HSP65 or phosphate-buffered saline (PBS). All animals were treated for 16 weeks. Reverse cholesterol efflux, the anti-oxidant and anti-inflammatory functions of HDL were assayed. Hepatocytes and peritoneal macrophages were isolated to examine the expression of cholesterol transport regulating proteins, including SR-B1, ABCA1, ABCG1, PPAR-γ and LXR-α. RESULTS In HSP65-immunized mice, paraoxonase1 (PON1) activity and the expression of IL-10 were reduced, while High-density lipoprotein inflammatory index (HII), myeloperoxidase (MPO) activity, and the expression of IFN-γ were elevated gradually. The MPO/PON1 ratio amount was significantly higher in HSP65-immunized group than in normal or PBS-immunized group. In addition, compared with normal or PBS-immunized group, cholesterol efflux rate and the expression of regulating proteins were markedly decreased in HSP65-immunized group. The mice immunized with HSP65 developed significantly larger aorta atherosclerotic plaques when compared with PBS-treated littermates. The high MPO/PON1 ratio was correlated with HII, cholesterol efflux rate and atherosclerotic plaques. CONCLUSIONS This study demonstrates that subcutaneous immunization with HSP65 impairs the properties of HDL, which may contribute to its important pathogenic role of HSP65 in atherogenesis. Also, MPO/PON1 ratio may be a predictor of AS.

Lck Inhibits Heat Shock Protein 65–Mediated Reverse Cholesterol Transport in T Cells

Previously, we reported that heat shock protein (HSP)65 impairs the effects of high-density lipoprotein on macrophages. We also showed that immune response activation adversely affects reverse cholesterol transport (RCT). In this study, we investigated the effects of the Src family kinase lymphocyte-specific protein tyrosine kinase (Lck) and elucidated the mechanism underlying HSP65-regulated cholesterol efflux in T cells. We evaluated cell proliferation, Lck expression, and inflammatory cytokine production in Jurkat cells and CD4+ T cells. HSP65-mediated inhibition of RCT was assessed by evaluating ABCA1, ABCG1, SR-BI, PPAR-γ, and liver X receptor-α expression. A dose-dependent relationship was found between the levels of these proteins and the suppression of cholesterol efflux. Stimulation of Lck-silenced T cells with ionomycin resulted in a decrease in intracellular calcium levels. Treatment of Jurkat cells with PP2, an inhibitor of Src family kinase, inhibited calcium-induced, but not PMA-induced, ERK phosphorylation. NF-κB activation in response to PMA was minimally inhibited in cells stimulated with PP2. HSP65 failed to trigger downstream ERK or JNK phosphorylation or to activate NF-κB or protein kinase C-γ in Lck-silenced cells. Additionally, elevation of intracellular calcium was also impaired. However, HSP65 significantly enhanced cholesterol efflux and decreased cellular cholesterol content by inducing the expression of cholesterol transport proteins in Lck-silenced cells. The treatment of Jurkat cells with PP2 also inhibited cell proliferation and promoted RCT. In conclusion, Lck is a key molecule in the TCR signaling cascade that inhibits cholesterol efflux and upregulates intracellular cholesterol ester content in T cells. Our results demonstrate that the immune response plays a previously unrecognized role in RCT.

Retarding the senescence of human vascular endothelial cells induced by hydrogen peroxide: effects of 17beta-estradiol (E2) mediated mitochondria protection.

This study investigates the influence of 17beta-estradiol (E2) on hydrogen peroxide (H2O2)-induced human vascular endothelial cell (HUVEC) senescence. HUVECs were divided into four groups, namely control group, H2O2 stimulation group, E2 intervention group and ICI182780 (ICI) intervention group. The aging-related β-galactosidase activities, cytochrome C oxidase activities, intracellular ATP levels, intracellular reactive oxygen species (ROS) levels and phosphorylated Rb protein expressions were mainly observed. Of which, senescence-associated β-galactosidase activities were detected using immunohistochemical staining, cytochrome C oxidase activities and intracellular ATP levels were detected using commercial kits, ROS levels were detected by fluorescence microscopy and fluorescence microplate reader, immunoblotting was used to quantitatively detect the expressions of phosphorylated Rb proteins. After continuous treatment of H2O2, the senescent phenotypes appeared in the HUVECs. The percentage of positive SA-βgal staining cells and the phosphorylated Rb expressions were significantly increased; intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels were elevated. Compared with the H2O2 stimulation group, E2 intervention significantly decreased the positive rate of SA-β-gal staining, the phosphorylated Rb protein levels, the intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels. Pretreatment of estrogen receptor blocker ICI182780 weakened the role of E2. These results indicated that H2O2 could induce HUVEC senescence; 17beta-E2 might relieve H2O2-induced mitochondrial damage through estrogen receptor and delay the vascular endothelial cell senescence.

Systematic analysis of the molecular mechanism underlying atherosclerosis using a text mining approach

BackgroundAtherosclerosis is one of the common health threats all over the world. It is a complex heritable disease that affects arterial blood vessels. Chronic inflammatory response plays an important role in atherogenesis. There has been little success in fully identifying functionally important genes in the pathogenesis of atherosclerosis.ResultsIn the present study, we performed a systematic analysis of atherosclerosis-related genes using text mining. We identified a total of 1312 genes. Gene ontology (GO) analysis revealed that a total of 35 terms exhibited significance (p < 0.05) as overrepresented terms, indicating that atherosclerosis invokes many genes with a wide range of different functions. Pathway analysis demonstrated that the most highly enriched pathway is the Toll-like receptor signaling pathway. Finally, through gene network analysis, we prioritized 48 genes using the hub gene method.ConclusionsOur study provides a valuable resource for the in-depth understanding of the mechanism underlying atherosclerosis.

The Impact of Serum Amyloid P-Component on Gene Expression in RAW264.7 Mouse Macrophages

Serum amyloid P-component (SAP) contributes to host defense and prevents fibrosis. Macrophages are the most abundant inflammatory cell type in atherosclerotic plaques. In the present study, using 3H-cholesterol-labeled counting radioactivity assay, we demonstrated that the apoAI-mediated cholesterol efflux in RAW264.7 macrophages was increased by SAP treatment in a time- and dose-dependent manner. We analyzed global gene expression changes upon SAP treatment using RNA sequencing. As a result, a total of 175 differentially expressed genes were identified, of which 134 genes were downregulated and 41 genes were upregulated in SAP treated cells compared to control cells. Quantitative RT-PCR analysis confirmed decreased expression of 5 genes and an increase in expression of 1 gene upon SAP treatment. Gene ontology analysis showed that genes involved in response to stimulus were significantly enriched in differentially expressed genes. Beyond protein-coding genes, we also identified 8 differentially expressed long noncoding RNAs. Our study may provide new insights into mechanisms underlying the functional role of SAP in macrophages.

Testosterone therapy delays cardiomyocyte aging via an androgen receptor-independent pathway

The testicular feminized (Tfm) mouse carries a nonfunctional androgen receptor (AR) and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1) and sham-operated Tfm mice (group 2, N = 8 each) received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7). Castrated littermates (group 4) and sham-operated untreated Tfm mice (group 5) were used as controls (N = 8 and 7, respectively). An additional control group (group 6) consisted of age-matched non-castrated littermates (N = 8). Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb) and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P < 0.01) and expression of p16INK4α, Rb and p53 proteins was significantly (P < 0.05) up-regulated in groups 4 and 5. These changes were improved to nearly normal levels in groups 1 and 2 (telomere length = 0.78 ± 0.05 and 0.80 ± 0.08; p16INK4α = 0.13 ± 0.03 and 0.15 ± 0.04; Rb = 0.45 ± 0.05 and 0.39 ± 0.06; p53 = 0.16 ± 0.04 and 0.13 ± 0.03), but did not differ between these two groups. These improvements were partly inhibited in group 3 compared with group 2 (telomere length = 0.65 ± 0.08 vs 0.80 ± 0.08, P = 0.021; p16INK4α = 0.28 ± 0.05 vs 0.15 ± 0.04, P = 0.047; Rb = 0.60 ± 0.06 vs 0.39 ± 0.06, P < 0.01; p53 = 0.34 ± 0.06 vs 0.13 ± 0.03, P = 0.004). In conclusion, testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.